Materials and methods relating to the diagnosis and treatment of diabetes and obesity

ABSTRACT

The diagnosis of diabetes based on the level or ratio of P- and A-type inositolphosphoglycans (IPGs) in a sample from a patient is disclosed.

This application is a 35 USC 371 of PCT/GB97/02440, filed Sep. 11, 1997.

FIELD OF THE INVENTION

The present invention relates to materials and methods for the diagnosisand treatment of diabetes and associated obesity.

BACKGROUND OF THE INVENTION

Diabetes is said to affect about 5% of the world population. Theetiology of the two major forms of diabetes, referred to asinsulin-dependent diabetes (IDDM) and non-insulin dependent diabetes(NIDDM), is quite different despite similarities in theirpathophysiologies. IDDM often arises in early life, and is due toautoimmune destruction of pancreatic β-cells resulting in partial orcomplete loss of β-cell function. NIDDM is more common in later life,typically more than 40 years in age, and about 85% of all diabetics havethis form. While the NIDDM group is best regarded as a heterogeneous setof disorders, two major sub-groups are recognised, these are thenon-obese, and the obese, with the latter being some 30% of the cases[1].

Some 60 years ago, Himsworth [2] first described the concept ofvariability in the sensitivity to insulin Insulin resistance, defined asthe impaired sensitivity of the effects of insulin on whole body glucoseutilization, has, as major components, the suppression of hepaticglucose production and the disposal of glucose loads by muscle viaglycogen synthesis and glucose oxidation [3,4]. The importance ofinsulin resistance in the pathophysiology of NIDDM and the increasedrisk factors for vascular disease in insulin resistant individuals hasbeen highlighted by Reaven [5,6], who described a cluster of riskfactors under the term 'syndrome X′ which included; glucose intolerance,high circulating insulin, disordered lipid metabolism and hypertension.The increased prevalence of NIDDM in developed countries together withits association with heart disease and stroke, makes this one of themost devastating diseases in the Western world.

It is, perhaps, surprising, despite the remarkable increase in ourunderstanding of the complex signalling functions of insulin, of thecellular and molecular mechanisms that underlie the diverse actions ofthis hormone, and of the role of mutant insulin receptors in insulinresistance, that standard texts and recent review state that “Theetiology and pathogenesis of the most frequent types of NIDDM, however,are not well defined” [1]; that “The mechanisms linking obesity andinsulin resistance are not known” [7]; and that “. . . the search forthe physiological, biochemical and molecular basis for cardiovascularrisk factor clustering in syndrome X continues” [8].

SUMMARY OF THE INVENTION

A new insight into the understanding of insulin action and NIDDM hasemerged from the identification and partial characterisation of twofamilies of inositol phosphoglycans (IPGs), each exhibiting specificinsulin-mimetic properties certain of which are shown in Table 1. TheIPG A-type stimulate lipogenesis, inhibit cAMP-dependent protein kinaseand modify the activity of adenylate cyclase and cAMP-phosphodiesterase,thus contributing to the control of cAMP and cAMP-regulatedintracellular processes which are classically inhibited by insulin. TheIPG P-type activate, among other enzymes, pyruvate dehydrogenasephosphatase (PDH P'ase), glycogen synthase phosphatase and glycerol3-phosphate acyl transferase. The activation of key phosphoproteinphosphatases plays a major role in the regulation of the disposal ofglucose by oxidative metabolism via PDH, and by the non-oxidative routeof storage by glycogen synthesis, both pathways classically known to beregulated by insulin [see 9-12].

The reported occurrence of inositol phosphoglycans in a wide range oftissues, and the influence of insulin on their release both in vivo andin vitro [10,12,13], has led to an intense interest in the role thesecompounds might play in the pathogenesis of experimental, genetic andclinical form of diabetes. Evidence that these inositol-containingcompounds are important in insulin signalling comes both from in vitrostudies on isolated cells and in vivo measurements using animal modelsof IDDM (type-I) and NIDDM (type-II) diabetes, including the findingsthat:

(a) Addition of antibody with anti-IPG specificity is able to block boththe metabolic and mitogenic actions of insulin [14, Rademacher et al,unpublished observations].

(b) Anti-IPG antiserum inhibits the stimulating effects of insulin andP-type IPG on adipocyte glycerol-3-phosphate acyltransferase in normalWistar rats [15].

(c) Mutant cells unable to synthesize IPGs respond to insulin asdetermined by tyrosine phosphorylation, but are not stimulated to elicitat least some of the metabolic effects of the hormone, in particularglycogen synthesis [16].

(d) The glycans promote serine/threonine dephosphorylation in adipocyteextracts via a mechanism requiring protein phosphatase 1, thephosphatase that regulates the activity of both glycogen synthase andphosphorylase [16,17].

(e) Impairment of glycosyl-phosphatidyl inositol-dependent insulinsignalling system in isolated rat hepatocytes by strepotozotocin-induceddiabetes [18].

(f) Diabetic GK rats, recognised as a model for insulin-resistant typeII diabetes [9], have a defect in synthesizing or releasing functionalIPGs as shown by the impaired insulin-induced activation ofglycerol-3-phosphate acyltransferase by a chiro-inositol-containinginsulin mediator [15] and impaired skeletal muscle glycogen synthaseactivation by insulin [19].

(g) Infusion of chiro-inositol into normal rats given a glucose load, orto streptozotocin-diabetic rats, results in decreased plasma glucose andenhanced activity of glycogen synthase: positive effects ofchiro-inositol treatment on insulin-resistant Rhesus monkeys have alsobeen reported [20,21].

Evidence that IPGs are important in the pathogenesis of humaninsulin-resistant type II diabetes derives largely from studies in whichtwo basic approaches have been used (see Table 2):

(a) Measurements of the free chiro- and myo-inositol content of urine ofdiabetic subjects using gas chromatography and mass spectrometry.

(b) Measurement of the bioactivity of inositol-phosphoglycan mediatorsin urine and tissue extracts employing bioassay procedures, e.g.activation of pyruvate dehydrogenase phosphatase, inhibition ofcAMP-dependent protein kinase. The main findings from these studies aregiven in Table 2.

In summary:

(a) Free chiro-inositol. This was shown to be decreased in urine ofNIDDM subjects by Kennington et al [22] and by Suzuki et al [23], and tobe decreased in urine of spontaneously diabetic rhesus monkeys [24]. Thedecreased urinary excretion rate has been reported to be directlyassociated with insulin resistance in a number of studies in humanpatients [22,25]. In contrast, increased urinary concentrations ofchiro-inositol were reported by Ostlune et al [26]. The discrepanciesbetween these reports have not been resolved.

(b) IPG P-type. Decreases in urinary excretion levels, as well asdecreased concentration of chiro-inositol-containing IPGs, were found inmuscle biopsy samples and haemodialysates of diabetic patients [25].

(c) Free myo-inositol. This was reported to be increased in urine ofNIDDM subjects in studies by Kennington et al [22] and by Ostlund et al[26].

(d) IPG A-type. Asplin et al [25] reported unchanged IPG A-Type in urineof NIDDM subjects using the bioassay system of inhibition ofcAMP-dependent protein kinase.

Two other lines of work give further support to the concept that IPGsplay a significant role in the insulin signal transduction system indiabetic patients.

(a) The report that increased plasma levels of chiro-inositol were foundin diabetic patients treated with insulin [Ostlund et al 26].

(b) Studies by Prochazha et al [27] on the genetic basis for insulinresistance in Pima Indians, centred on the genetic analysis of proteinphosphatase 1 (PP1), a key regulatory enzyme in glycogen synthesis.Their conclusion that the structural gene for PP1 catalytic b subunitdoes not appear to be a major genetic determinant responsible for PP1abnormalities, lends further support to the concept of a key role forinositol-containing phosphoglycans in the aetiology of insulinresistance and disordered glycogen metabolism.

In view of: (i) the growing body of evidence pointing to the importanceof the inositol phosphoglycans in insulin action and disorders ofinsulin response in NIDDM, (ii) the key role IPG P-type and IPG A-typeplay in the regulation of enzymes involved in disposal of glucose byoxidative and non-oxidative routes, in the regulation of lipogenesis,triacylglycerol formation and lipolysis, and in glaconecogenesis, (iii)the divergent and meagre data on the bioactive species of IPG P-type andIPG A-type in urine; it was deemed important to make a detailed study ofthe relationship between urinary IPGs and NIDDM in a cross-sectionalstudy of spot urine samples from a randomised series of male diabeticpatients, (iv) studies on the measurement in urine of chiro andmyo-inositol have been complicated by the fact that both breakdown ofendogenous IPGs and dietary sources of the sugars will be present. Thus,prior art studies in this area which assumed that the P-type mediatorcontains chiro-inositol and that the A-type mediator containsmyo-inositol must be interpreted with caution, see Fonteles, M C, Huang,L C, Larner, J, Diabetologia, 39:731-734, (1996), in which the authorsreport that they incorrectly identified the inositol in the P-typemediator which is pinitol and not chiro-inositol. As pinitol is notconverted to chiro-inositol by the acid conditions used in carbohydrateanalysis, this is an example of misidentification in this area.

This invention arises from the discovery, as detailed below, of acorrelation between levels of A-type and P-type IPGs, and the ratio ofP-type to A-type, and the occurrence of certain forms of diabetes andobesity.

The present invention provides, inter alia:

(1) Diagnosis of type II diabetes (NIDDM) by measuring the ratio ofP-type:A-type mediators in blood or urine.

(2) Treatment of IDDM or lean type II diabetes (NIDDM) (BMI<27) with amixture of P- and A-type mediators.

(3) Treatment of obese type II diabetes (NIDDM) with a P-type mediatorand/or an A-type antagonist.

A therapeutic treatment for type II obese diabetics comprisesadministering an antagonist to A-type IPG. We show herein (see FIG. 5B)that male obese type II diabetics release a 2:1 unit ratio of P:Amediators. The A-type drives glucose into fat while the P-type drivesglucose into glycogen for energy consumption. In the type II diabetics,insulin stimulates the release of 6 units of P-type for every 3 units ofA-type. In contrast for lean diabetics or type I (insulin deficient) orcontrol patients insulin stimulates the release of 6 units of P-type forevery one unit of A-type. The obese diabetics therefore drive threetimes as much glucose into fat as the normal controls or lean diabetics.80% of blood glucose is normally taken up by muscle which is a P-typeresponsive tissue. In the type II diabetics, less goes into muscle andmore into fat, but removal by fatty tissue is less efficient andconsequently the blood glucose levels rise, giving rise to the diabeticstate. These patients normally are then hyperinsulinaemic which pushesmore glucose into the fat compounding the problem and setting up avicious cycle.

For treatment of type I diabetes, about a 6:1 mixture of P:A mediatorscan be used in males, and about a 4:1 mixture in females.

Accordingly, in a first aspect, the present invention provides a methodof diagnosing diabetes, the method comprising determining the level orratio of P- and/or A-type inositolphosphoglycans (IPGs) in a biologicalsample from a patient. The determination of this ratio helps toaccurately assign the patient to a diabetic group, allowing thetreatment of diabetes in the patient to be tailored accordingly to thatgroup and/or the patient's individual needs, e.g. by then administeringto the patient appropriate amounts of P- or A-type IPGs, or theirantagonists, to correct the levels and/or ratio of these compounds inthe patient.

In one embodiment, the method of diagnosing diabetes comprises the stepsof:

(a) contacting a biological sample obtained from the patient with asolid support having immobilised thereon a first binding agent havingbinding sites specific for one or more P-type IPGs and a second bindingagent having binding sites for one or more A-type IPGs;

(b) contacting the solid support with one or more labelled developingagents capable of binding to unoccupied binding sites, bound IPGs oroccupied binding sites; and,

(c) detecting the label of the developing agents specifically binding instep (b) to obtain values representative of the levels of the P- andA-type IPGs in the sample.

Preferably, the method comprises the further step of:

(d) using the values to obtain a ratio of the P- and A-type IPGs in thesample.

Additionally or alternatively, the levels of the P- and/or A-type IPGsin a sample, and hence their ratio, can be determined using one or morethe assays for P- and A-type biological activity described below.

In a further aspect, the present invention provides the use of P- orA-type inositolphosphoglycans (IPGs), or antagonists to P- or A-typeIPGs, in the preparation of a medicament for the treatment of diabetes.

In one embodiment, the present invention provides the use of an A-typeIPG antagonist and/or a P-type IPG in the preparation of a medicamentfor the treatment of obese type II diabetes. As described above, thesepatients have a form of diabetes characterised by an reduced ratio ofP:A-type IPGs.

In a further embodiment, the present invention provides the use ofP-type and A-type IPGs in the preparation of a medicament for thetreatment of IDDM or lean type II diabetes (NIDDM). In this embodiment,preferably, the P:A-type ratio is about 6:1 mixture for male patientsand about a 4:1 mixture for female patients. In this use, the P- andA-type IPGs can be administered to the patient separately or formulatedtogether for administration. As mentioned above, it is expected thatformulations will be tailored to each individual patient depending onthe levels or ratio of the IPGs in the patient. The formulations may betailored by the physician or by the patient at the time ofadministration.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising an A-type IPG antagonist and/or a P-type IPG incombination with a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a mixture of P- and A-type IPGs in combinationwith a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a kit for treatingobese type II diabetes comprising a first container of P-type IPG and asecond container of A-type IPG antagonist, for simultaneous orsequential administration.

In a further aspect, the present invention provides a method ofscreening for P- or A-type IPG antagonists, the method comprising:

(a) contacting a candidate antagonist and a P- or A-type IPG in an assayfor a biological property of the P- or A-type IPG under conditions inwhich the IPG and the candidate antagonist can compete;

(b) measuring the biological property of the IPG; and,

(c) selecting candidate antagonists which reduce the biological activityof the IPG.

The present invention will now be described by way of example and not bylimitation with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a graph of IPG A- and IPG P-type content of urine fromdiabetic subjects (arranged in order of IPG A-type content).

FIG. 1B shows a graph of IPG A- and IPG P-type content of urine fromcontrol subjects (arranged in order of IPG A-type content).

FIG. 2 shows the IPG A- and IPG P-type content of urine from diabeticsubjects showing high IPG A-type and high IPG P-type groups with theirHbA1 values.

FIGS. 3A,B shows the correlation between IPG A-type and IPG P-type andHbA1.

FIGS. 4A,B shows the correlation between IPG A-type and IPG B-type andbody mass index.

FIG. 5 shows (A) the relationship between IPG A-type and IPG B-type andbody mass index in diabetic subjects, (B) the ratio of IPG A-type:IPGB-type as a function of body mass index, and insulin sensitivity (Mvalue)as a function of body mass index. In (A), the diabetic subjectswere divided into 7 groups according to their body mass index(BMI−kgm²). The average values for the IPG A-type and the IPG P-type foreach subgroup are shown. The average values for the IPGs of the controlgroup are shown by vertical columns. The BMI of the control group was24±2.4 (mean±SD). The arrows and numbers along the abscissa show thenumber of diabetic subjects in each group receiving either insulintreatment or metformin with or without other treatments. Metformin isthe treatment of choice for overweight and obese NIDDM subjects. In (B)the vertical dotted line marks the conventional cut-off for thedefinition of obesity (BMI>27).

FIGS. 6A,B shows the relationship between systolic blood pressure andurinary IPG A- and P-types in diabetic (IDDM and NIDDM) subjects.

DETAILED DESCRIPTION OF THE INVENTION IPGS

Studies have shown that A-type mediators modulate the activity of anumber of insulin-dependent enzymes such as cAMP dependent proteinkinase (inhibits), adenylate cyclase (inhibits) and cAMPphospho-diesterases (stimulates). In contrast, P-type mediators modulatethe activity of insulin-dependent enzymes such as pyruvate dehydrogenasephosphatase (stimulates) and glycogen synthase phosphatase (stimulates).The A-type mediators mimic the lipogenic activity of insulin onadipocytes, whereas the P-type mediators mimic the glycogenic activityof insulin on muscle. Both A- and P-type mediators are mitogenic whenadded to fibroblasts in serum free media. The ability of the mediatorsto stimulate fibroblast proliferation is enhanced if the cells aretransfected with the EGF-receptor. A-type mediators can stimulate cellproliferation in the chick cochleovestibular ganglia.

Soluble IPG fractions having A-type and P-type activity have beenobtained from a variety of animal tissues including rat tissues (liver,kidney, muscle brain, adipose, heart) and bovine liver. A- and P-typeIPG biological activity has also been detected in human liver andplacenta, malaria parasitized RBC and mycobacteria. The ability of ananti-inositolglycan antibody to inhibit insulin action on humanplacental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPGaction on rat diaphragm and chick ganglia suggests cross-speciesconservation of many structural features. However, it is important tonote that although the prior art includes these reports of A- and P-typeIPG activity in some biological fractions, the purification orcharacterisation of the agents responsible for the activity is notdisclosed.

In co-pending patent applications claiming priority from GB-A-9618930.3and GB-A-9618929.5, we have described the isolation and characterisationof P-type and A-type IPGs.

A-type substances are cyclitol-containing carbohydrates, also containingZn²⁺ ion and optionally phosphate and having the properties ofregulating lipogenic activity and inhibiting cAMP dependent proteinkinase. They may also inhibit adenylate cyclase, be mitogenic when addedto EGF-transfected fibroblasts in serum free medium, and stimulatelipogenesis in adipocytes.

P-type substances are cyclitol-containing carbohydrates, also containingMn²⁺ and/or Zn²⁺ ions and optionally phosphate and having the propertiesof regulating glycogen metabolism and activating pyruvate dehydrogenasephosphatase. They may also stimulate the activity of glycogen synthasephosphatase, be mitogenic when added to fibroblasts in serum freemedium, and stimulate pyruvate dehydrogenase phosphatase.

In view of the optional presence of phosphate in the A- and P-type IPGs,references to “inositolphosphoglycans” or “IPGs” include compounds inwhich phosphate is not present. These compounds are alternatively betermed inositolglycans (IGs).

The A- and P-type substances were also found to have the followingproperties:

1. Migrates near the origin in descending paper chromatography using4/1/1 butanol/ethanol/water as a solvent.

2. The substances contains phosphate which is directly related toactivity.

3. The free GPI precursors are resistant to cleavage by GPI-PLC.

4. They are bound on Dowex AG50 (H+) cation exchange resin.

5. They are bound on an AG3A anion exchange resin.

6. The activity is resistant to pronase.

7. They are detected using a Dionex chromagraphy system.

8. The P-type substance is partially retained on C-18 affinity resin.

The A- and P-type substances may be obtained from human liver orplacenta by:

(a) making an extract by heat and acid treatment of a liver homogenate,the homogenate being processed from tissue immediately frozen in liquidnitrogen;

(b) after centrifugation and charcoal treatment, allowing the resultingsolution to interact overnight with an AG1-X8 (formate form) anionexchange resin;

(c) collecting a fraction having A-type IPG activity obtained by elutingthe column with 50 mM HCl, or a fraction having P-type IPG activityobtained by eluting the column with 10 mM HCl;

(d) neutralising to pH 4 (not to exceed pH 7.8) and lyophilising thefraction to isolate the substance.

(e) descending paper chromatography using 4/1/1 butanol/ethanol/water assolvent.

(f) purification using high-voltage paper electrophoresis inpyridine/acetic acid/water.

(g) purification using Dionex anion exchange chromatography, orpurification and isolation using Vydac 301 PLX575 HPLC chromatography.

More details of the methods for obtaining these IPGs are provided in thesaid patent applications, the contents of which are incorporated hereinby reference.

Antagonists

As mentioned above, P-type or A-type IPG antagonists include substanceswhich have one or more of the following properties:

(a) substances capable of inhibiting release of the P- or A-typemediators;

(b) substances capable of reducing the levels of P- or A-type IPG via anIPG binding substance (e.g. an antibody or specific binding protein);and/or,

(c) substances capable of reducing the effects of P- or A-type IPGs.

In one embodiment, the IPG antagonists are specific binding proteins.Naturally occurring specific binding proteins can be obtained byscreening biological samples for proteins that bind to IPGs.

In a further embodiment, the antagonists are antibodies capable ofspecifically binding to P- or A-type IPGs. The production of polyclonaland monoclonal antibodies is well established in the art. Monoclonalantibodies can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP-A-184187, GB-A-2188638or EP-A-239400. A hybridoma producing a monoclonal antibody may besubject to genetic mutation or other changes, which may or may not alterthe binding specificity of antibodies produced.

Antibodies may be obtained using techniques which are standard in theart. Methods of producing antibodies include immunising a mammal (e.g.mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al, Nature, 357:80-82, 1992). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith any of the proteins (or fragments), or may be one constructed usingsequences obtained from an organism which has been exposed to theantigen of interest.

Antibodies according to the present invention may be modified in anumber of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or otherbinding partner are the Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

Humanised antibodies in which CDRs from a non-human source are graftedonto human framework regions, typically with the alteration of some ofthe framework amino acid residues, to provide antibodies which are lessimmunogenic than the parent non-human antibodies, are also includedwithin the present invention

A hybridoma producing a monoclonal antibody according to the presentinvention may be subject to genetic mutation or other changes. It willfurther be understood by those skilled in the art that a monoclonalantibody can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP-A-184187, GB-A-2188638or EP-A-0239400. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

Hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells, eukaryotic or prokaryotic, containing nucleic acid encodingantibodies (including antibody fragments) and capable of theirexpression. The invention also provides methods of production of theantibodies including growing a cell capable of producing the antibodyunder conditions in which the antibody is produced, and preferablysecreted.

The antibodies described above may also be employed in the diagnosticaspects of the invention by tagging them with a label or reportermolecule which can directly or indirectly generate detectable, andpreferably measurable, signals. The linkage of reporter molecules may bedirectly or indirectly, covalently, e.g. via a peptide bond ornon-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

In a further embodiment, the IPG antagonists are synthetic compounds.These may be produced by conventional chemical techniques or usingcombinatorial chemistry, and then screened for IPG antagonist activity.These compounds may be useful in themselves or may be used in the designof mimetics, providing candidate lead compounds for development aspharmaceuticals. Synthetic compounds might be desirable where they arecomparatively easy to synthesize or where they have properties that makethem suitable for administration as pharmaceuticals, e.g. antagonistwhich are peptides may be unsuitable active agents for oral compositionsif they are degraded by proteases in the alimentary canal. Mimeticdesign, synthesis and testing is generally used to avoid randomlyscreening large number of molecules for a target property.

Production of Monoclonal Antibodies

Inositolphosphoglycan (IPG) purified from rat liver by sequential thinlayer chromatography (TLC) was used to immunize New Zealand rabbits andBalb/c mice by using conventional procedures.

After immunisation, monoclonal antibodies were prepared using theapproach of fusion of mouse splenocytes (5×10⁶ cells/ml) with mutantmyeloma cells (10⁶ cells/ml). The myeloma cell lines used were thoselacking hypoxanthine-guanine phosphoribasyl transferase. The screeningmethod of hybridoma cells was based on a non-competitive solid-phaseenzyme immunoassay in which the antigen (IPG) was immobilised on a solidphase. Culture supernatant were added and positive hybridoma cells wereselected.

A single cell cloning was made by limiting dilution.

Hybridomas for three monoclonal antibodies (2D1, 5HG and 2P7) wereselected. All monoclonal antibodies were determined to be IgM using aEK-5050 kit (Hyclone).

In order to test that all monoclonal antibodies recognised IPGs, anon-competitive solid-phase enzyme immunoassay was used. F96 PolysorpNunc-Immuno Plates are used for the assay. The polysorp surface isrecommended for assays where certain antigens are immobilised.

The immobilised antigen (IPG) diluted to 1:800 captured the monoclonalantibody from tissue culture supernatant, ascitic fluid, and when thepurified monoclonal antibody was used.

The detection method used an anti-mouse IgM, biotinylated whole antibody(from goat) and a streptavidin-biotinylated horseradish peroxidasecomplex (Amersham), ABTS and buffer for ABTS (Boehringer Mannheim).

The same immunoassay was used to evaluate the polyclonal antibody. Inthis assay, the detection method employed an anti-rabbit Ig,biotinylated species—specific whole antibody (from donkey).

The antibodies can be purified using the following method. Fast ProteinLiquid Chromatography (Pharmacia FPLC system) with a gradient programmerGP-250 Plus and high precision pump P-500 was used in order to purify apolyclonal IPG antibody.

A HiTrap protein A affinity column was used for purification ofpolyclonal IPG from rabbit serum. Protein quantitation was made using aMicro BCA protein assay reagent kit (Pierce).

Monoclonal IgM antibodies were purified in two steps. Ammonium sulfateprecipitation was the method chosen as a first step. Tissue culturesupernatant was treated with ammonium sulfate (50% saturation). Pelletdiluted in PBS was transferred to dialysis tubing before the secondstep.

Since ammonium sulfate precipitation is not suitable for a single steppurification, it was followed by gel filtration chromatography-antibodysolution in PBS run into a Pharmacia Sepharose 4B column. Proteinquantitation was made reading the absorbance at 220-280 nm in aPerkin-Elmer lambda 2 UV/VIS spectrophotometer.

Protocol for Sandwich ELISA

The protocol below sets out an indirect, non-competitive, solid-phaseenzyme immunoassay (sandwich ELISA) for the quantification ofinositolphosphoglycans (IPG) in biological fluids, such as human serum.

In the assay, monoclonal IgM antibodies are immobilised on a solidphase. Tissue culture supernatant, ascitic fluid from mice with aperitoneal tumour induced by injecting hybridoma cells into theperitoneum and purified monoclonal antibody have been used in theimmunoassay. F96 Maxisorp Nunc-Immuno plates were used for these assays.Maxisorp surface is recommended where proteins, specially glycoproteinssuch as antibodies, are bound to the plastic.

The immobilised antibody captures the antigen from the test sample(human serum or IPG used like a positive control).

A bridging antibody (a purified polyclonal IPG antibody from rabbit) isneeded to link the anti-antibody biotinylated to the antigen.

The detection method employs an anti-rabbit Ig, biotinylatedspecies-specific whole antibody (from donkey) and astreptavidin-biotinylated horseradish peroxidase complex (Amersham),ARTS and buffer for ABTS (Boehringer Mannheim).

The ELISA assay can be carried out as follows:

1. Add 100 μl/well in all the steps.

2. Add monoclonal antibody diluted 1:100 in PBS in a F96 MaxisorpNunc-Immuno plate. Incubate at least 2 days at 4° C.

3. Wash with PBS three times.

4. Add a blocking reagent for ELISA (Boehringer Mannheim) in distilledwater (1:9) 2 hours at room temperature.

5. Wash with PBS-Tween 20 (0,1%) three times.

6. Add a purified polyclonal antibody (diluted 1:100 in PBS), overnightat 4° C.

7. Wash with PBS-Tween 20 (0.1%) three times.

8. Add an anti-rabbit Ig, biotinylated species-specific whole antibody(from donkey) (Amersham) diluted 1:1000 in PBS, 1 h 30 min at roomtemperature.

9. Wash with PBS-Tween 20 (0.1%) three times.

10. Add a streptavidin-biotinylated horseradish peroxidase complex(Amersham) diluted 1:500 in PBS, 1 h 30 min at room temperature.

11. Wash with PBS three times.

12. Add 2.2-Azino-di-(3-ethylbenzthiazoline sulfonate (6)) diammoniumsalt crystals (ABTS) (Boehringer Mannheim) to buffer for ABTS (BM):Buffer for ABTS is added to distilled water (1:9 v/v). 1 mg of ABTS isadded to 1 ml of diluted buffer for ABTS.

13. Read the absorbance in a Multiscan Plus P 2.01 using a 405 mm filterin 5-15 min.

Pharmaceutical Compositions

The mediators and antagonists of the invention can be formulated inpharmaceutical compositions. These compositions may comprise, inaddition to one or more of the mediators or antagonists, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nture of the carrier or other materialmay depend on the route of administration, e.g. oral, intravenous,cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Whether it is a polypeptide, antibody, peptide, small molecule or otherpharmaceutically useful compound according to the present invention thatis to be given to an individual, administration is preferably in a“prophylactically effective amount” or a “therapeutically effectiveamount” (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Diagnostic Methods

Methods for determining the concentration of analytes in biologicalsamples from individuals are well known in the art and can be employedin the context of the present invention to determine the ratio of P- andA-type inositolphosphoglycans (IPGs) in a biological sample from apatient. This in turn can allow a physician to determine which of thesub-groups of diabetes a patient suffers from, and so optimise thetreatment of it, in the case of type II obese diabetics, avoiding orameliorating the symptoms of syndrome X. Examples of diagnostic methodsare described in the experimental section below.

Preferred diagnostic methods rely on the determination of the ratio ofP- and A-type IPGs. The methods can employ biological samples such asblood, serum, tissue samples or urine.

The assay methods for determining the concentration of P- and A-typeIPGs typically employ binding agents having binding sites capable ofspecifically binding to one or more of the P or A-type IPGs inpreference to other molecules. Examples of binding agents includeantibodies, receptors and other molecules capable of specificallybinding the IPGs. Conveniently, the binding agent(s) are immobilised onsolid support, e.g. at defined locations, to make them easy tomanipulate during the assay.

The sample is generally contacted with the binding agent(s) underappropriate conditions so that P-and A-type IPCs present in the samplecan bind to the binding agent(s). The fractional occupancy of thebinding sites of the binding agent(s) can then be determined using adeveloping agent or agents. Typically, the developing agents arelabelled (e.g. with radioactive, fluorescent or enzyme labels) so thatthey can be detected using techniques well known in the art. Thus,radioactive labels can be detected using a scintillation counter orother radiation counting device, fluorescent labels using a laser andconfocal microscope, and enzyme labels by the action of an enzyme labelon a substrate, typically to produce a colour change. The developingagent(s) can be used in a competitive method in which the developingagent competes with the analyte for occupied binding sites of thebinding agent, or non-competitive method, in which the labelleddeveloping agent binds analyte bound by the binding agent or to occupiedbinding sites. Both methods provide an indication of the number of thebinding sites occupied by the analyte, and hence the concentration ofthe analyte in the sample, e.g. by comparison with standards obtainedusing samples containing known concentrations of the analyte. Inpreferred embodiments, this can then be used to determine the P:A typeratio.

A. Objectives

In view of the potential importance of inositol phosphoglycans,(i) tothe understanding insulin signal transduction systems, (ii) to theaetiology of insulin resistance, NIDDM and syndrome X, (iii) as a markerfor the early detection of insulin-resistant diabetes, and (iv) as apotential therapeutic agent in NIDDM, a study has been undertaken tomeasure the urinary levels of inositol phosphoglycans in normal malesubjects and in male diabetic patients, including IDDM, NIDDM (both leanand obese), using bioassay procedures in which the stimulation of wellestablished systems, known to be activated by these insulin-mediators,were employed (see Experimental section below).

The concentration and ratio IPG P-type and IPG A-type in urine sampleswere determined, and comparison made with clinical data at theUniversity College, London Hospitals, with a view to gaining informationon the possible links between changes in IPGs and markers of the typeand severity of diabetes. The clinical parameters included: HbA₁,urinary creatinine and protein, blood pressure, body mass index (weightkg/height m²), complications (e.g. cardiovascular, renal, retinal,neurological), age and treatment. The duration of treatment at thehospital is known, but firm evidence of the total duration of diabetesis not always available or reliable, and has not been included here.

This cross-sectional study of a randomised selection of 30 diabeticsubjects and approximately the same number of non-diabetic control malesubjects will not reveal whether any correlations observed betweenclinical markers and changes in IPGs represent a cause, a consequence ora coincidence. Nevertheless, it is anticipated that the present datawill provide:

(a) Additional information on the concentration of and direction ofchange of the bioactive urinary IPGs in diabetic male subjects—a matterof some controversy at this time;

(b) New information leading to a better understanding the biochemicalbasis of syndrome X;

(c) A starting point for consideration of the rationale of treatment ofinsulin resistant diabetics with IPGs.

B. Experimental

1. Assay of IPG A-type and IPG P-type Activity:

The activity of P- and A-type IPGs in urine extracts were studied usingspecific bioassay procedures. IPG P-type was determined using theactivation of PDH phosphatase [28]. The PDH complex and PDH phosphatase(metal-dependent form) were prepared from beef heart as described byLilley et al [28] and the assay of the activation of the phosphatase wasperformed by the spectrophotometric variant of the two-stage systemdescribed by these authors. This assay is considered to be acharacteristic feature of IPG P-type (see Larner et al [29]). IPG A-typewas determined by the stimulation of lipogenesis as measured by theincorporation of [U-¹⁴C] glucose into the lipids of adipocytes isolatedfrom epididymal fat pads by the method of Rodbell [30]. A high degree ofspecificity for IPG A-type was found for this bioassay.

A straight line relationship between added IPGs and the stimulation ofPDH phosphatase activity (IPG P-type) and lipogenesis in intactadipocytes (IPG A-type) was obtained; this relationship held at least upto a stimulation of +250%. These observations provided a basis for aunit to be defined and used for the purpose of comparison of yields ofIPGs from different tissues and urine samples. Linearity between IPGadded and the percentage change in response, has been observed by others(see Lilley et al [28] and Newman et al [31]), although Asplin et al[25] did not show linearity in their study on IPGs in human urine fromnormal and diabetic subjects, an effect which was particularly markedwith the IPG A-type.

2. Extraction of IPG P-type and IPG A-type From Urine:

Urines were extracted as described by Asplin et al [25]. The finalfractions were freeze dried and stored at −20° C. For use, the IPGfractions were resuspended in is water, immediately before assay, sothat 10 μl of redissolved IPG corresponded to 10 ml urine. In view ofthe possibility that high, and varying, amounts of IPGs might beexcreted in the different groups of subjects, and in order to ensurethat the capacity of the resin was well in excess of the load applied,preliminary test runs were made to determine the optimal ratio of resinto starting urine volume. Linearity of recovery was obtained up to 100ml urine per 18 g resin. In the present study, the ratio of 50 ml urineto 19 g resin Has maintained to allow for variation in IPG content.

3. Expression of Results:

A unit of IPG is defined as the amount causing a 50% activation in thebasal level of the test system.

The yield of IPGs in urine is given on two different bases:

(i) Percentage stimulation of the test system by 10 μl final urineextract, allowing direct comparison with data of Asplin et al [15].

(ii) Units of IPG per 1 mmol creatinine.

The results are given as means±SEM, and as median values with the rangeof values.

4. Design of Experiment:

A cross-sectional study was undertaken with random spot samplescollected at outpatients clinics. The patients included NIDDM subjectscontrolled by diet alone, by sulphonylureas, by metformin, by insulin ora combination of any of these treatments. Only a limited number of IDDMsubjects were available. In the present survey, male subjects were used,this avoided complications arising from varying hormone profiles inwomen of different ages, in particular with or without HRT orcontraceptive pills. The avoidance of a mix of male/female surveys wasdeemed important, since a separate study in this laboratory revealedthat the IPG P-type/IPG A-type ratio was significantly lower urine fromwomen than from men, largely Is- related to their higher IPG A-typeconcentration (see Table 3A). In addition to the measurement of IPGP-type and IPG A-type in urine the following clinical data was madeavailable:

Urine: Creatinine, Protein

Blood: HbA1

Biodata: Age, weight and height (for calculation of BMI), bloodpressure, complications (eg. cardiovascular, renal, neuropathy), ethnicorigin.

Treatment: Insulin, sulphonyl ureas, biguanides, diet, (singly orcombined).

The changes in inositol phosphoglycans in diabetes were correlated withthe degree of glycaemic control as shown by HbA1, with obesity asindicated by basal metabolic index (BMI), with age and with bloodpressure.

C. Results

1. Inositol Phosphoglycans in Urine of Diabetic and Control Subjects:

The concentration of IPG P-type and IPG A-type in urine of male diabeticsubjects and non-diabetic controls are shown in Table 3A. The resultsare given as means±SEM and as median values together with their range.The most significant differences, taking the diabetic group as acomposite whole, are the rise in IPG A-type and the unchanged IPG P-typein urine relative to the control group. These differences aresignificant both as concentration per ml urine and as units per mmolcreatinine; the ration IPG P-type/IPG A-type fell in the diabetic group.The biodata relating to the diabetic and control subject is shown inTable 3B.

The present results contrast with those of Asplin et al [25], whoreported, on the basis of a much smaller study, that IPG A-type wasunchanged in NIDDM diabetes while IPG P-type decreased. These authorsnoted a non-linearity in their experiments in the dose response curvefor IPG P-type (pH 2.0 fraction) measured by the stimulation of PDHphosphatase; such problems were not encountered in the study reportedhere. A further difference was in the bioassay systems used to measureIPG A-type, Asplin et al [25] employed the inhibition of cAMP-dependentprotein kinase, wile the: present report is based on activation oflipogenesis by IPG A-type in isolated adipocytes.

Table 3A also includes data on the concentration of IPGs in the urine ofnon-pregnant women, a control group taken from a separate study on theurinary IPGs in pregnancy in normal, pre-eclamptic and diabetic women.The higher value for IPG A-type and for the lower value of the IPGP-type/IPG A-ratio for women relative to control male subjects arehighly significant; in contrast the IPG P-type is substantially the samein both groups. These data underline the importance of separateassessment of the urinary IPGS in men and women in studying changes indiabetes (c.f. reports in [22], [25], [32]).

The heterogeneity of the underlying causes of diabetes, particularly inNIDDM [1,4,33-35], led to the re-examination of the data on the diabeticsubjects to determine whether:

(a) There was a constant or varying relationship between the IPG A- andP types in diabetic subjects.

(b) Any correlation could be found between IPG profiles and markers ofthe degree of glycaemic control (HbA1) obesity, and hypertension, allfactors of significance in relation to syndrome X.

(c) There was any correlation between treatments and IPG profile.

2. Relationships Between IPG A- and P-types in Diabetic Subjects:

Our first approach, in view of the highly significant change found inIPG A-type in urine of diabetic subjects, was to examine the results indescending order of the magnitude of this A-type (see FIG. 1A). It isclear that there are major differences across the series, both in theconcentration of the A and P-types and in their ratios. Further, itappeared that there was a rough reciprocal relationship between thesemediators, and that sub-groups could be distinguished which displayedeither a high IPC, A-type/IPG P-type ratio or, conversely, had a highIPG P-type/IPG A-type ratio. There was a gradation across the series,and these extreme groups were separated by an intermediate group. Thecomparable data for the control group, similarly presented, is shown inFIG. 1B. It is clear that the control group has a narrower range of IPGconcentration and presents as a more homogeneous sample without theextremes of high IPG A- or high IPG P-types seen in the diabetic groups.It is notable that the control subjects approximate to the intermediategroup seen in the diabetic profile (FIG. 1A).

The differentiation of sub-groups within the diabetic subjects isfurther emphasised by outlining those values for IPG A-type or IPGP-type which are greater than values observed in the relativelyhomogeneous control non-diabetic group. These are shown, boxed in, forthose subjects where the urinary IPG A-type or IPG P-type content isabove the maximum control value (FIG. 2). The diabetic sub-groups sodefined, as high IPG A-group and high IPG P-group, were considered torepresent subjects outside the norm and to be worthy of specialexamination.

3. Correlation Between Markers of Diabetic Status and IPGs:

3.1 Relationship Between HbA1 and High IPG A—High IPG P-groups ofDiabetics:

High values of HbA1, indicative of a sustained high is blood glucoselevels, may be taken, as a first approximation, to be the net effect ofglucose intake, hepatic production by gluconeogenesis and whole bodyutilization, and thus to be an index of glucose intolerance, a featureof syndrome X.

When the correlations between HbA1 and urinary concentrations of IPGA-type and IPG P-type were examined, a positive correlation was shownbetween HbA1 and urinary IPG A-type, contrasting with the negativecorrelation with IPG P-type; these relationships are shown in FIGS.3A,B. The high IPG A-group was associated with a significantly raisedHbA1 value of 11.6±0.5 (mean±SEM) while the high IPG P-group had a lowermean HbA1 value of 10.0±0.3, the difference between these two groups wassignificant (P<0.01) (see FIG. 2). From these results, it is suggestedthat diabetics with a high IPG A-type in the urine may exhibit onefeature of syndrome X, namely glucose intolerance and a related insulinresistance. In contrast, diabetic subjects with a high IPG P value and alower value for HbA1 might have a more effective rate of disposal ofglucose via oxidative routes involving pyruvate dehydrogenase andstorage via glycogen synthesis, and/or a decreased hepatic glucoseproduction, all systems known to be regulated in part by IPG-type, thushaving a better regulation of blood glucose, less intolerance to glucoseand, in parallel, a lower HbA1.

3.2 Relationship Between Obesity (BMI) and IPG A-and P-types inDiabetes:

Two major subgroups of NIDDM are recognised, these are the obese andnon-obese, with the latter being some 30% of the cases [1]. It was,therefore, of interest to examine whether the urinary IPGs in diabeticsubjects showed any correlation with the degree of obesity as evaluatedby the calculated body mass index. Subjects with values of BMI above 27are held to be overweight, those over 30 to be obese [36].

There was a strong negative correlation between urinary IPG P-type andBMI in diabetic subjects (P,<0.01) and a positive correlation with IPGA-type (FIGS. 4A,B). The profile of change is more clearly seen whengroupings of 4 or 5 individuals, having closely similar BMI values, areshown, together with the concentration of urinary IPG A- and P-typesand, in particular, when the data are presented as BMI versus the ratioof IPG P-type/IPG A-type. These results are presented in FIGS. 5A,B.

The most striking feature of these graphs is that lean subjects have ahigh IPG P-type and a low IPG A-type in urine, while the reverse is truefor the obese subjects. Also significant is the observation that thecurve for each of the IPGs cross-over at a value of the BMI about 27,the figure above which the clinicians consider a patient is overweight,patients with a BMI of 30 or more are classified as obese.

As indicated in FIG. 5A, 8 out of 9 patients treated with insulin,including the IDDM group and 3 NIDDM patients receiving insulin, fellwithin the normal or lean segment below the cut-off at a BMI value of27. The majority of diabetic patients with a BMI value above 27 werereceiving treatment with metformin, with or without other drugs or diet;this reflects the preferred treatment of overweight or obese diabeticpatients with the biguanide, metformin [37,38].

The clinical significance of the present results given 5 in FIG. 5A isfurther emphasised when compared with data from the literature [36],also shown in FIG. 5B. The survey reported by Ferrannini [36] comparesthe rate of glucose utilization by muscle to BMI in normal and obesepatients, and show the well-established cross-over at 27, clearlyrelating a depressed rate of glucose utilization to obesity. Linkingdata from these two figures (FIGS. 5A,B), it is postulated that the rateof glucose utilization by muscle is related to the IPG P- to A-typeratio. This leads to the important conclusion that the degree of Isglucose intolerance in obese NIDDM subjects may be directly related tothe profile of IPGs, a low P-type being associated with both obesity andless well controlled blood glucose values.

3.3 Relationship Between Hypertension and IPG A- and P-types inDiabetes:

The individual values for systolic blood pressure versus theconcentration of the two types of IPG are shown in FIGS. 6A,B, fromwhich it can be seen that those subjects with the highest blood pressurehad the lowest IPG P-value, while those with a normal systolic bloodpressure, of around 120, had IPG P values within the normal range (FIG.6A). Conversely, there is a positive correlation between IPG A andsystolic pressure (FIG. 6B). Both correlations are significant.

Since systolic blood pressure is correlated both with age and, as shownin the present study, with urinary IPG P-type, it was necessary todemonstrate that age was not a confounding factor in the IPGP-type/blood pressure association. An analysis by simple partialcorrelation demonstrated that there was a significant correlationbetween IPG P-type and blood pressure independent of the age factor.

D. Discussion

1. Urinary Levels of Bioactive Forms of IPGs in Diabetes:

The present cross-sectional survey of the bioactive forms of inositolphosphoglycans in the urine of male IDDM and NIDDM patients advancesknowledge of the relationships between IPGs and clinical diabetes byproviding evidence for an association between IPG P-type and IPG A-typeconcentrations and ratios, and degree of glycaemic control, systolicblood pressure, obesity and treatment. Differences between male andfemale non-diabetic control subjects were also recorded.

A significant correlation between urinary chiro-inositol excretion andin vivo insulin resistance has been reported in clinical surveys[32,40], in type II diabetic Rhesus monkeys [22] and in the GK rat modelof type II diabetes [23]. The association between insulin resistance andobesity has also been clearly established [36]. Thus, the presentobservation that a low IPG-P/IPG-A ratio, is associated with obesity inNIDDM patients is entirely in keeping with the concept of the importanceof IPG A-type and obesity. The differences between the present data andreported surveys [32] emphasise the importance of the use of biologicalassay systems to determine the links between inositol phosphoglycans andpathophysiological changes.

In a recent review Craig et al [32], stated that there was nosignificant correlation between glycated haemoglobin concentrations andurinary concentrations of chiro-inositol in a group of type II diabeticpatients as a whole. The present work demonstrates a significantpositive correlation between HbA1 and IPG A-type, and a negativecorrelation with IPG P-type in urine (FIGS. 3A,B). The boxed data inFIG. 2 highlights the group with an association of a high IPG-A/IPG-Pratio and poor metabolic control as revealed by HbA1.

It must be stressed that it may be even more important in attempting torelate changes in this putative mediator to diabetic status, todetermine urinary IPG A-type by a bioassay system, rather then bymyo-inositol excretion data, since myo-inositol is a product of theglucuronic acid cycle in kidney, a metabolic route known to be increasedin experimental diabetes in the kidney and this would be furtherenhanced by renal growth in early diabetes [41].

If the relationships between obesity, raised HbA1 (values>11) and a lowIPG P-type and/or low IPG-P/IPG-A ratio are confirmed in a moreextensive study, then a basis for initial screening of patients withpossible deficiencies in this putative second messenger system will havebeen identified, based on routine clinical monitoring of patients onattendance at clinic.

2. Inositol Phosthoglycans and Syndrome X:

The correlations shown between urinary IPG P-type and IPG A-type andHbA1, obesity and blood pressure in diabetic subjects provides a basisfor speculation on the links between inositol phosphoglycans andsyndrome X in NIDDM (Table 4). Any such speculation starts with thepremise that the concentration of urinary IPGs are an indicator ofcirculating levels of these insulin mediators, and, thus that a lowurinary IPG-P/IPG-A, or conversely high IPG-A/IPG-P, ratio is mirroredin the plasma levels and indicates the changes in the milieu interieurto which organs and tissues are exposed.

In summary, as shown in Table 5, section A, it is proposed that theswitch over form the high P to A ratio in normal subjects and leandiabetics to a low P/A ratio in obese NIDDM patients is a criticalfactor in the obese syndrome. The expected effect of a low IPG P-type onaspects of glucose metabolism, based on the known effects of thisputative insulin mediator on enzyme systems, is shown in section B.These include: (i) a decrease in glucose conversion to glycogen, (ii) adepressed pyruvate oxidation, and (iii) an increased hepatic productionof glucose and (iv) increased hepatic glucose 6-phosphatase. Together,these changes will depress the ability of muscle to dispose of a glucoseload, a major disturbance as muscle normally accounts for some 70% ofglucose utilisation. This effect, together with a failure to suppresshepatic gluconeogenesis and glucose 6-phosphatase, would contribute toelevated blood glucose levels and glucose intolerance, key features ofsyndrome X.

The parallel effect of a high prevailing IPG A-type would be to compoundthe effects of the low IPG P-type described above (Table 5, section C).Firstly, IPG A-type stimulates lipogenesis in adipocytes and activatesacetyl CoA carboxylase, thus increasing lipid synthesis. is Secondly, byinhibition of hormone-induced lipolysis, as a resultant effect ofdamping down the cAMP response, a raised IPG A-type would be expected todrive the balance between lipogenesis and lipolysis towards lipidsynthesis and storage, contributing to obesity and disordered lipidmetabolism, again a feature of syndrome X. An ancillary factor in thedyslipidemias of insulin-resistant NIDDM individuals may reside in thediminished adrenal function in diabetes [42], an effect which may belinked to altered cAMP regulation.

The role of IPGs in activating phosphatases involved in the regulationof pyruvate dehydrogenase, and glycogen synthesis IPG P-type, and in theregulation of cAMP linked systems, via the inhibition of cAMP-dependentprotein kinase and adenylate cyclase, and activation of low Km cAMPphosphodiesterase, thus exerting a controlling influence onhormone-induced cAMP accumulation [10,43,44] via IPG A-type, placesthese putative second messengers or mediators at the heart of metabolicregulation in the cyclic processes of proteinphosphorylation/dephosphorylation, constant cycling being of primeimportance as a background to rapid hormone response [45].

The present observations that there is a strong negative correlationbetween the concentration IPG P-type in urine and systolic bloodpressure, and a significant, but less marked, positive correlation withIPG A-type (FIGS. 6A,B), deserve more detailed consideration in thelight of the importance of hypertension as a factor in syndrome X [5,6],and the fact that approximately 40% of individuals with NIDDM arehypertensive and have an increased risk of cardiovascular disease [46].

The question arises as to the manner in which the IPGs might link,directly or indirectly, with systems influencing blood pressure.Derangements in NIDDM in two major systems linked to regulation of bloodpressure have been reviewed recently, these are the role of thesympathoadrenal system [42], and the potential role of theendothelium-derived nitric oxide system (EDNO) [47]. An involvement ofIPG P-type in the generation of NO by endothelial cells would be aparticularly attractive hypothesis.

Baron [47] has provided evidence for the linking the EDNO system ininsulin action which rests on the observations that:

(a) Insulin produces a specific increase in blood flow in skeletalmuscle.

(b) Insulin stimulation of glucose uptake by muscle is associated withincreased vasodilation.

(c) That insulin mediated vasodilation occurs by the release of NO asindicated by the use of inhibitors of NO synthase activity.

(d) The effect of insulin on the dose response curve for methacholine(an acetylcholine-type compound which cause the synthesis and release ofEDNO), is shifted to the left, consistent with insulin modulation thesynthesis/release of EDNO. Evidence suggesting that insulin causes anincrease in the production of EDNO in insulin sensitive individuals, butnot in insulin resistant subjects, led to the proposal that theendothelium is an insulin target tissue [47].

(e) Thus, insulin resistance in obesity may be at the level of therelease of NO by endothelial cells, leading to impaired vasodilation thepresence of insulin. Such an impairment would, in turn, result in anassociated reduction in the rate of insulin-mediated glucose uptake, andto enhanced pressor sensitivity [47]. Such an hypothesis would link themajor aspects of syndrome X to the ubiquitous nitric oxide signallingsystem [48].

The question of the role played by IPG P-type in the sequence postulatedby Baron [47] remains open to question, although the role of IPG P-typeas a putative insulin second messenger, together with the present data,strongly suggests that IPGs are part of this integrated system. Theassociation of manganese with IPG P-type, is of interest in thiscontext, not only for the requirement for manganese by the guanylatecyclase system, associated with the generation of cGMP in the smoothmuscle relaxant signal initiated by NO, but also for the adducts formedbetween manganese and NO [49], suggesting a possible role for IPG P-typein the transport and/or sequestering of this cellular signallingmolecule, with, perhaps, special reference to the regulation of proteinphosphatase-1 [50].

The present data, taken in conjunction with new concepts un theimportance of endothelial cell function in pathophysiological states,focuses attention on the possible significance of IPGs as centralfactors in the aetiology of syndrome X.

3. Aspects of the Use of Insulin and IPGs in Treatment of NIDDM:

The treatment of diabetic patients is a subject continuously underreview, perhaps most marked for those with NIDDM; a number ofprospective studies have been undertaken to assess the best regime [see38]. With respect to treatment by injection of insulin, criticismsinclude the fact that this means of administration results in highperipheral insulin levels, possibly contributing to abnormalities inlipid metabolism and vascular complications, the physiological route ofrelease into the portal circulation and delivery to the liver beingby-passed [51]. Recent studies by Kubot et al [52] have providedevidence demonstrating the superiority of portal insulin delivery onportally loaded glucose handling over peripheral deliver, a matter whichhas been the subject of some controversy [see 52]. The oraladministration of IPGs would have the potential advantage of portaldelivery, thus approaching a more normal physiological relationshipbetween the putative mediators of insulin action and the disposal of aningested glucose load.

Secondly, when relatively high levels of insulin are administered toachieve an acceptable level of blood glucose control, as in severeinsulin resistance, there is the danger of inappropriate stimulation oftissues by cross-reactivity of insulin with IGF-I receptors [53]. Theappearance of skin lesions of acanthosis nigricans and ovarian cellthecal hyperplasia with hyperandrogenism are reported to be associatedwith insulin resistance; theca cells possess both insulin and IGF-Ireceptors which can signal increased steroidogenesis [53]. O'Rahilly &Moller reported that although insulin receptor mutations are uncommon inpatients with typical NIDDM, there is a practical problem of treatingindividuals with severe insulin resistance associated with suchmutations, and they cite the use of IGF-I as a treatment for some cases.

For several decades a family of drugs, the sulphonylureas, has been amajor therapeutic agent in the treatment of NIDDM; and, indeed, in theUSA when the biguanide, phenformin, was withdrawn from the market [54],sulphonylureas were the only type of compound available for thetreatment of NIDDM, although recently the biguanide metformin wasapproved by the Food and Drug Administration for use in the USA [55].The metabolic disturbances of NIDDM are widely held to be the resultanteffect of an interaction between insulin resistance and impaired insulinsecretion [55,56]. There is evidence that the sulphonylureas act both incausing an acute stimulation of the rate of insulin release from thepancreas, and in increasing glucose uptake and utilization byextrapancreatic tissues, with evidence for sulphonylureas enhancingbasal and insulin-stimulated glucose transport and metabolism in muscleand adipose tissue in animals and in humans (see Muller et al [57]). Theobservation that a sulphonylurea drug, glimpiride, stimulates therelease of glycosyphosphatidlyinositol-anchored plasma-membrane proteinsfrom 3T3 adipocytes was of particular interest in the present context,since this potent insulin-mimetic drug appeared to have common featureswith the putative insulin second messenger system of the inositolphosphoglycans [9-15 12,58]. It is important to note, however, thatglimpirides have not been shown to induce the cleavage of free GPIs, theIPG precursors.

This evidence for a possible common locus of action between insulin anda sulphonylurea in releasing a precursor of inositol phosphoglycan fromcell membranes reinforces, in some measure, the view that inositolphosphoglycans have a potential for therapeutic use in the treatment ofNIDDM. The importance of the present invention in suggesting treatmentof diabetes with IPGs per se, as an alternative to using a drugliberating IPG precursors, is perhaps indicated by the statement in Arecent review that “Patients with overt NIDDM have reduced responses tomany insulin secretagogues, including glucose and non-glucose stimuli,such as sulphonyulureas, arginine, and leucine. Patients treated withsulphonylureas have high primary and secondary failure rates.” [56].Thus, based on the study here, inositol phosphoglycans, and/or theirprecursors, might be more effective in patients failing to respond fullyto treatment with sulphonylureas by normalisation of blood glucose.

The biguanide, metformin is the treatment widely chosen for treatment ofdiabetic patients with NIDM, insulin resistance and obesity [37]. Whilethis drug reduces fasting glucose levels and reduces hepatic glucoseproduction [37,55], it clearly fails to restore the pattern of excretionof IPG P-type to normal, this marker remaining significantly belowcontrol values in the present survey (FIGS. 5A,B). The apparent failureof metformin to restore the IPG P-type to normal values may have abearing on the lactic acidosis found with this class of compound, seenmost notably with the biguanide, phenformin, now withdrawn. While lacticacidosis is a relatively minor problem when metformin is given toselected patients free from hepatic or renal disease, it remains afactor to be considered as a potential hazard in some individuals[37,55]. On the basis of the present work, it is suggested that aninappropriately low IPG P-type might be associated with a diminishedactivity of PDH phosphatase and an associated alteration in the activeinactive forms of PDH, leading to the diversion of pyruvate generated inthe glycolytic pathway to lactic acid.

On the basis of current problems with conventional treatments, some ofwhich are outlined, there would appear to be a case for considering theuse of IPGs or of precursors or of antagonists of the compounds, as anadjunct in the treatment of diabetes. Among the positive aspects of theuse of IPGs are the small molecular weight and heat and acid stability;thus, these compounds should be suitable for oral administration, anddelivery to the live, via the hepatic portal circulation, might avoidsome of the problems associated with peripheral tissue overload withinsulin [51,52].

It is probable that a fine balance exists between IPG P-type and IPGA-type, which preliminary data suggests may be tissue specific inresponse to hormone stimuli [13]. The knowledge of the structure andfunction of these second messengers offers a new avenue to theunderstanding and treatment of diabetes.

TABLE 1 Some Properties of Inositol Phosphoglycans IPG P-Type IPG A-TypeCHEMICAL COMPONENTS Cyclitol Cyclitol Carbohydrates CarbohydratesPhosphate Phosphate Metal (Mn²⁺/Zn²⁺) Metal (Zn²⁺) SEPARATION AG 1-x8 pH2.0 pH 1.3 BIOACTIVITY Activation of: Pyruvate DH P'ase LipogenesisGlycogen synthase Acetyl CoA carbox. Low-km cAMP phosphodiesteraseInhibition of: cAMP - PK cAMP - PK G6P phosphatase Adenylate cyclaseMETABOLIC EFFECTS Increased glucose Increased lipid utilisation byLowers cAMP - muscle via counteracting oxidation (PDH) & effect ofglycogen synthesis catecholamines e.g. (70% of glucose on lipolysis loadused by muscle) ——————————— ↓ Jointly promote dephosphorylation ofenzymes regulated by phosphorylation- dephosphorylation cycle

TABLE 2 Reported changes in free Chiro- and Myo-Inositol in urine ofNIDDM subjects (estimated by GC/MS of derivatised product)Chiro-inositol Decreased in urine of NIDDM subjects Normal 89: NIDDM 1.8umol/day [22] Normal 96: NIDDM 32 umol/day [23] Increased in urine ofNIDDM subjects Normal 2.1: NIDDM 12 umol/day [26] Myo-inositol Increasedin urine of NIDDM subjects Normal 176: NIDDM 444 umol/day [22] Normal86: NIDDM 825 umol/day [26] Reported changes in mediator activity of IPGP-Types and IPG A-Types in urine of NIDDM subjects (estimated bybioassay - PDH P'ase, cAMP PK) IPG P-Type (chiro-inositol containing)Decreased in urine of NIDDM subjects [25] IPG A-Type (myo-inositolcontaining) Unchanged in urine of NIDDM subjects [25] (Note: only 9controls and 4 NIDDM subjects, assays non- linear) [22] Kennington etal. 1990; [23] Suzuki et al. 1994; [25] Asplin et al. 1993; [26] Ostlundet al. 1993

TABLE 3a The IPG A-type and IPG P-type content of urine from normal anddiabetic (IDDM and NIDDM) male subjects. IPG A-TYPE IPG P-TYPE IPG-P IPGA-type IPG P-type CREATININE GROUP (% stimulation by 10 ml urine) IPG-A(Units/mmol creatinine) (mmol/L) URINARY IPG A-TYPE AND IPG P-TYPE INGROUPS OF DIABETIC AND CONTROL MALE SUBJECTS VALUES EXPRESSED AS MEANS ±SEM CONTROLS (27) 29.2 ± 3.7 89.3 ± 8.3  3.06 7.86 ± 1.56 21.6 ± 2.549.86 ± 0.93 DIABETICS (30)  67.5 ± 11.1 98.6 ± 12.1 1.46 24.7 ± 4.8 31.1 ± 4.5  7.37 ± 0.56 Fisher's P <0.01 NS <0.01 NS <0.05 VALUESEXPRESSED AS MEDIANS AND RANGE CONTROLS (27) 25 (71-5) 82 (172-7) 4.2(15.8-0.2) 4.88 (33.2-1.0) 16.8 (62.1-8.3) 8.5 (20.8-3.2) DIABETICS (30)48 (265-2) 81 (308-30) 1.3 (18-0.2) 13.5 (107-3.1) 25.2 (139-6.6) 6.6(11.75-2.8) Mann-Whitney test NS <0.001 NS <0.05 URINARY IPG A-TYPE ANDIPG P-TYPE IN A GROUP OF NON-DIABETIC FEMALE SUBJECTS VALUES EXPRESSEDAS MEANS ± SEM CONTROL FEMALE (10) 997.8 ± 11.8 68.2 ± 12.3 0.70 32.7 ±6.02 18.8 ± 1.96 7.11 ± 0.81 Fisher's P (males v females) <0.001 NS<0.001 NS <0.05

TABLE 3b Biodata - Control and Diabetic Subjects CONTROLS DIABETICS (Nondiabetic) (IDDM & NIDDM) No. of subjects 27 30 Age (years) 51.5 ± 14.659.7 ± 12.2 (29-69) (28-82) Body mass index 24.7 ± 2.4  28.0 ± 3.9 (22.3-30.8) (21.9-38.5) Creatinine (mmol/L) 9.85 ± 4.6  7.19 ± 3.0 (3.24-20.8) (2.84-15.6) HbA1 Not measured 10.8 ± 1.6  (Normal range 5-8) (8.5-14.7) BP systolic Not measured 139 ± 15  (111-172) BP diastolicNot measured 80 ± 9  (57-97) Ethnic origin 25/2 19/11 Caucasian/AsianTreatment Insulin alone — 6 Insulin ± another — 3 Metformin ± another —11 Sulphonylurea ± — 10 another Values are given as means ± SD with therange shown in parentheses.

TABLE 4 Insulin Resistance and Obesity. “The mechanisms linking obesityand insulin resistance are not known” [Walker. Obesity, insulinresistance and its link to NIDDM. 1995] At least 30% of NIDDM patientsare obese (body mass index BMI > 30) and are at increased risk orcardiovascular disease. The cluster of risk factors “syndrome X” Insulinresistance Glucose intolerance High circulating insulin Disordered lipidmetabolism Hypertension [Reavan. Role of insulin resistance in humandisease. 1988; Walker, 1995]

TABLE 5 A CHARACTERISTICS OF OBESE NIDDM SUBJECTS AND CHANGES IN IPG P-AND A-TYPES OBESITY Low P High A RAISED HbA1 Low P High A (Obesity +raised HbA1 indicates glucose intolerance) HIGH BLOOD Low P High APRESSURE B EFFECT OF LOW IPG P-TYPE ON METABOLIC PATHWAYS GLUCOSEGLYCOGEN Decreased PYRUVATE Decreased Glucose OXIDATION intoleranceHEPATIC GLUCOSE Increased PRODUCTION C EFFECT OF HIGH IPG A-TYPE ONMETABOLIC PATHWAYS LIPOGENESIS Increased ACETYL CoA Increased Increasedfat CARBOX. RAISED cAMP Decreased synthesis and LIPOLYSIS Decreasedstorage

References:

1. Bennett, P. H., Bogardus, C., Tuomilehto, L. and Zimmet, P. 1992.Epidemiology and natural history of NIDDM: Non-obese and obese. In:International Textbook of Diabetes mellitus. Eds. Alberti, K. G. M. M.,DeFronzo, R. A., Keen, H. and Zimmet, P. pp147-169. John Wiley & SonsLtd.

2. Himsworth, H. P. 1936. Diabetes mellitus: its differentiation intoinsulin-sensitive and insulin insensitive types. Lancet i. 127-130.

3. DeFronzo, R. A. 1988. The Triumvirate: beta cell, muscle, liver: acollusion responsible for NIDDM. Diabetes, 37: 667-687.

4. DeFronzo, R. A., Bonadonna, R. C. and Ferrannini, E. 1992.Pathogensis of NIDDM. A balanced overview. Diabetes Care, 15: 318-368.

5. Reaven, G. M. 1988. Banting Lecture. Role of insulin resistance inhuman disease. Diabetes, 37: 1595-1607.

6. Reaven, G. M. 1995. Pathophysiology of insulin resistance in humandisease. Physiol. Rev. 75: 473-486.

7. Walker, M. 1995. Obesity, insulin resistance, and its link tonon-insulin-dependent diabetes mellitus. Metabolism, 44 (Suppl. 3):18-20. p b 8. Williams, B. 1994. Insulin resistance: the shape of thingsto come. Lancet, 344: 521-524.

9. Larner, J., Huang, L. C., Tang, G., Susuki, S., Schwartz, C. F. M.,Romero, G., Roulidis, Z., Zeller, K., Shen, T. W., Oswald, A. S., andLutterell, L. 1998. Insulin Mediators: Structure and Formation. ColdSprings Harbor Symp. 53: 965-971.

10. Romero, G., and Larner, J., 1993. Insulin Mediators and theMechanism of Insulin Action. Adv. Pharm. 24: 21-50.

11. Romero, G., 1991. Inositol glycans and cellular signalling. CellBiology International reports. 15:827-852.

12. Rademacher, T. W., Caro. H., Kunjara. S., Wang, D. Y., Greenbaum, A.L. and McLean, P. 1994. Inositolphosphoglycan second messengers.Brazilian J. Med. Biol. 27:327-341.

13. Kunjara, S., Caro, H. N., McLean, P. and Rademacher, T. W. 1995.Tissue specific release of inositol phosphoglycans. In Svasti, J. et al(Eds). Biopolymers and bioproducts: Structure, function andapplications. Bangkok, Thailand. Samakkhisan (Dokya). Public Co. Ltd.301-306.

14. Romero, G., Gamez, G., Huang, L. C., Lilley, K., and Lutterell, L.1990. Antiinositolglycan antibodies selectively block some of theactions of insulin in intact BC3H1 cells. Proc. Natl. Acad. Sci. USA.87: 1476-1480.

15. Varese, R. V., Standaert, M. C., Yamada, K., Huang, C., Zhang, C.,Cooper, D. R., Wang, Z., Yang, Y., Susuki, S., Toyota, T. and Larner, J.1994. Insulin-induced activation of glycerol 3-phosphate acyltransferaseby chiro-inositol-containing insulin mediator is defective in adipocytesof insulin resistant, type II diabetic Goto-Kakizaki rats. Proc. Natl.Acad. Sci. 91: 11040-11044.

16. Lazar, D. F., Knez, J. J., Medoff, M. E., Cuatracasa, P., andSaltiel, A. P. 1994. Stimulation of glycogen synthesis by insulin inhuman erythroleukemia cells requires the synthesis ofglycosyl-phosphatidylinositol Proc. Natl. Acad. Sci. USA. 91:9665-9669.

17. Misek, D. E., and Saltiel, A. R. 1994. An inositol phosphate glycanderived from a Trypanosoma brucei glycosyl phosphatidylinositol promotesprotein dephosphorylation in rat epidiymal adipocytes. Endocrinology,135: 1869-1876.

18. Sanchez-Arias, J. A., Sanchez-Gutierrez, J. C., Guadano, A.,Alvarez, J. F., Samper, B., Mato, J. M. and Feliu, J. E. 1992.Impairment of glycosyl-phosphatidylinositol-dependent insulin signalingsystem in isolated rat hepatocytes by streptozotocin-induced diabetes.Endocrinology, 131:1727-1733.

19. Villar-Palasi, C., and Farese, R. V. 1994. Impaired skeletal muscleglycogen synthase activation by insulin in the Goto-Kakizaki (G/K) rat.Diabetologia, 37: 885-888.

20. Ortmeyer, H. K., Huang, L. C., Zhang, L., Hansen, B. C., and Larner,J. 1993. Chiro-inositol deficiency and insulin resistance. II. Acuteeffects of D-chiro-inositol administration in streptozotocin-diabeticrats given a gloucose load, and spontaneously insulin resistance Rhesusmonkeys. Endocrinology, 132: 646-651.

21. Huang, L. C., Fonteles, M. C., Houston, D. B., Hang, C., and Larner,J. 1993. Chiro-inositol deficiency and insulin resistance. III Acuteglycogenic and hypoglycaemic effects of two inositol phosphoglycaninsulin mediators in normal and streptozotocin-diabetic rats in vivo.Endocrinology, 132: 652-657.

22. Kennington, A. S., Hill, C. H., Craig, J., Bogardus, C., Raz, I.,Ortmeyer, H. K., Hansen, B. C., Romero, G., and Larner, J. 1990. Lowurinary chiro-inositol excretion in non-insulin dependent diabetesmellitus. New Engl. J. Med. 323: 373-378.

23. Suzuki, S., Tanada, Y., Hirai, S., Abe, S., Sosaki, A., Suzuki, K.,Toyata, T. 1991. In: New Directions in Research and Clinical Works forObesity and Diabetes mellitus. Eds. Angei, A., Hotta, N. pp 197-203.Elsevier.

24. Ortmeyer, H. K., Bodkin, N. L., Lilley, K, Larner, J. and Hanson, B.C. 1993. Chiro-inositol deficiency and insulin resistance inspontaneously diabetic Rhesus monkeys. Endocrinology, 132, 640-645.

25. Asplin, I., Galasko, G., and Larner, J. 1993. chiro-Inositoldeficiency and insulin resistance: A comparison of the chiro-inositol-and the myo-inositol-containing insulin mediators isolated from urine,hemodialysate, and muscle of control and type II diabetic subjects.Proc. Natl. Acad. Sci. USA. 90: 5924-5928.

26: Ostlund, R. E., McGill, J. B., Herskowitz, I., Kipnis, D. M.,Santiago, J. V., and Sherman, W. R. 1993 D-chiro-inositol metabolism indiabetes mellitus. Proc. Natl. Acad. Sci. USA. 90: 9988-9992.

27. Prochazka, M., Mochizuki, H., Baier, L. J., Cohen, P. T. W., andBogardus, C. 1995. Molecular and linkage analysis of type-1 proteinphosphatase catalytic b subunit gene: lack of evidence for its majorrole in insulin resistance in Pima Indians. Diabetologia, 38: 461-466.

28. Lilley, K., Zhang, C. L., Villar-Palasi, C., Larner, J., and Huang,L. 1992. Insulin mediator stimulation of pyruvate dehydrogenasephosphatase, Arch. Biochem. Biophys. 296: 170-174.

29. Larner, J., Huang, L. C., Suzuki, S., Tang, E., Zhang, C., Schwartz,C. F. W., Romero, G., Luttrell, L. and Kennington, A. S. 1989. Insulinmediators and the control of pyruvate dehydrogenase complex. Annals N.Y.Acad. Sci. 573: 297-305.

30. Rodbell, M. 1964. Metabolism of isolated fat cells. J. Biol. Chem.239: 375-380.

31. Newman, J. D., Armstrong, J., McD., and Bornstein, J. 1985. Assay ofinsulin mediator activity with soluble pyruvate dehydrogenasephosphatase. Endocrinology 116: 1912-1919.

32. Craig, J. W., Larner, J. and Asplin, C. M. 1994. Chiroinositoldeficiency and insulin resistance. In. Molecular Biology of Diabetes.Part II. Eds. Draznin, B. and LeRoith, D., humana Press Inc. Totowa. NJ.

33. Serrano, J., Mateo, C. M. and Caro, J. F. 1992. Insulin resistance:cellular and molecular mechanisms. In: recent Advances in Endocrinologyand Metabolism. Vol. 4: pp. 167-183.

34. Moller, D. E. and Flier, J. S. 1991. Insulin resistance—mechanisms,syndrome, and implications, New Engl. J. Med. 325: 938-948.

35. Krentz, A. J. and Nattrass, M. 1996. Insulin resistance: amultifaceted metabolic syndrome. Insights gained using a low-doseinsulin infusion technique. Diabetic Medicine, 13: 30-39.

36. Ferrannini, E. 1995. Physiological and metabolic consequences ofobesity. Metabolism, 44: (Suppl.3) 15-17.

37. DeFronzo, R. A. and Goodman, A. N. and the Multicenter MetforminStudy Group 1995. Efficacy of metformin in patients with non-insulindependent diabetes mekkitus. New Engl. J. Med. 333: 541-549.

38. United Kingdom Prospective Diabetes Study (UKPDS). 13: Relativeefficacy of randomly allocated diet, sulphonylurea, insulin or metforminin patients with newly diagnosed diabetes followed for three years.1995. B,M. J. 310: (6972): 83-88.

39. Snedecor, G. W. 1964. Statistical methods. Fifth Edition. Iowa StateUniversity Press, Ames Iowa, USA.

40. Suzuki, S., Kawasaki, H., Satoh, Y., Ohtomo, M., Hirai, M., Hirai,A., Hirai, S., Onoda, M., Matsumoto, M., Hirokio, Y., Akai, H., Craig,J., Larner, J. and Toyota, T. 1994. Urinary chiro-inositol excretion isan index marker of insulin sensitivity in Japanese Type II diabetes.Diabetes Care.

41. Sochor, M., Baquer, N. Z. and McLean, P. 1985. Glucose over-andunder-utilization in diabetes. Comparative studies of changes inactivities of enzymes of glucose metabolism in rat kidney and liver.Molecular Physiol. 2: 51-68.

42. Reaven, G. M., Lithel, H. and Landsberg, L. 1996. Hypertension andassociated metabolic abnormralitia—the role of insulin resistance andthe sympathoadrenal system. New Engl. J. Med. 334: 374-381.

43. Machicao, F., Mushack, J., Seffer, E., Ermel, B. and Haring, H. U.1990: Mannose, glucosamine and inositol monophosphate inhibit theeffects of insulin on lipogenesis. Further evidence for a role forinositol oligosaccharides in insulin action. Biochem. J. 266: 909-916.

44. Martiny, L., Antonicelli, F., Thuillez, B., Lambert, B., Jacquemin,C., and Haye, B. 1990: Control by thyotropin of the production bythyroid cells of an inositol phosphate-glycan. Cell Signalling 2: 21-27.

45. Brautigan, D. L 1994. Protein phosphatases. Recent Prog. HormoneRes. 49: 197-214.

46. Panzram, G., 1987. Mortality and survival in type(non-insulin-dependent) diabetes mellitus. Diabetologia, 30: 123-131.

47. Baron, A. D. 1996. The coupling of glucose metabolism and perfusionin human skeletal muscle. The potential role of endothelium-derivednitric oxide. Diabetes, 45 (Suppl. 1): S105-S109.

48. Moncada, S. and Higgs, A. 1993. Mechanisms of disease: the1-arginine nitric oxide pathway. New Engl. J. Med. 329: 2002-2012.

49. Cotten, F. A. and Wilkinson, G. 1972. Advanced Inorganic Chemistry.Third edition, Interscience publishers. New York, London.

50. Cohen, P. 1989. The structure and regulation of proteinphosphatases. Annu. Rev. Biochem. 58: 453-508.

51. Alberti K. G. M. M. and Press, C. M. 1982. The Biochemistry of theComplications of Diabetes Mellitus. pp 231-270. Eds Keen, H. and Jarret,J. Publishers Edware Arnold Ltd, London.

52. Kubota, M., Yamasaki, Y., Sekiya, M., Kubota, M., Morishima, T.,Kishimoto, R., Shichiri, M and Kamada, T. 1996. Portal insulin deliveryis superior to peripheral delivery in handling of portally deliveredglucose. Metabolism, 45: 150-154.

53. O'Rahilly, S. and Moller, D. E. 1992. Mutant insulin receptors insyndromes of insulin resistance. Clinical Endocrinology, 36: 121-132.

54. Williams, R. H. and Palmer, J. P. 1975. Farewell to phenformin fortreating diabetes mellitus. Ann. Intern. Med. 83: 567-568.

55. Sturnvoll, M., Nurjhan, N., Perriello, G., Dailey, G. and Gerich, J.E. 1995. Metabolic effects of metformin in non-insulin-dependentdiabetes mellitus. New Engl. J. Med. 333: 550-554.

56. Polansky, K. S., Sturis, J. and Bell, G. I. 1996.Non-insulin-dependent diabetes mellitus—a genetically programmed failureof the beta cell to compensate for insulin resistance. New Engl. J. Med.334: 777-783.

57. Muller, G., Dearey, E. A. and Punter, J. 1993. The sulphonylureadrug, glimepiride, stimulates release ofglycosylphosphatidylinositol-anchored plasma-membrane proteins from 3T3adipocytes. Biochem. J. 289: 509-521.

58. Romero. G., Lutterll, A., Rogol, A., Zeller, K., Hewlett, E. andLarner, J. 1988. Phosphatidylinositol-glycan anchors of membraneproteins; potential precursors of insulin mediators. Science (Wash. DC)240: 509-512.

What is claimed is:
 1. A method of diagnosing diabetes, the methodcomprising determining: (a) the level of P- or A-typeinositolphosphoglycans (IPGs), (b) the ratio of P-type to A-type IPGs,or (c) the ratio of A-type to P-type IPGs in a biological sample from apatient, wherein: an increased level of A-type IPGs, a reduced P-type toA-type IPG ratio, or an increased A-type to P-type IPG ratio, ascompared to a control level or ratio, is indicative of obese type IIdiabetes; or an increased level of P-type IPGs, an increased P-type toA-type IPG ratio, or a reduced A-type to P-type IPG ratio, as comparedto a control ratio, is indicative of lean type II diabetes; and thelevel of P-type IPGs is determined using an assay selected from thegroup consisting of measurement of activation of pyruvate dehydrogenasephosphatase and an immunoassay, and the level of A-type IPGs isdetermined using an assay selected from the group consisting ofmeasurement of activation of lipogenesis in isolated adipocytes and animmunoassay.
 2. The method of claim 1 wherein the biological sample is ablood or urine same.
 3. The method of claim 1 wherein the level of theP- or A-type IPGs is determined using an assay measuring activation ofpyruvate dehydrogenase phosphatase and/or activation of lipogenesis inisolated adipocytes, respectively.
 4. The method of claim 3 wherein thelevel of the P-type IPGs is determined in an assay measuring activationof pyruvate dehydrogenase phosphatase by P-type IPGs.
 5. The method ofclaim 3 wherein the level of the A-type IPGs is determined in an assaymeasuring activation of lipogenesis by A-type IPGs in isolatedadipocytes.
 6. The method of claim 1 wherein the level of the P- and/orA-type IPGs is determined using an immunoassay.
 7. The method of claim 6wherein the level of the P-type IPGs is determined using an immunoassay.8. The method of claim 1 comprising: (a) contacting a biological sampleobtained from the patient with a solid support having immobilisedthereon a first antibody having one or more binding sites specific forone or more P-type IPGs and a second antibody having one or more bindingsites specific for one or more A-type IPGs; (b) contacting the solidsupport with labelled developing agents capable of binding to IPGs boundto antibodies or capable of binding to anti-IPG antibodies; and, (c)detecting the label of the developing agents specifically binding in (b)to obtain values representative of the levels of the P- and A-type IPGsin the sample.
 9. The method of claim 6 wherein wherein the level of theA-type IPGs is determined using an immunoassay.
 10. The method of claim6 wherein the biological sample is a blood or urine sample.
 11. Themethod of claim 8 wherein the biological sample is a blood or urinesample.
 12. The method of claim 8 wherein at least one labelleddeveloping agent is capable of binding to bound IPGs.
 13. The method ofclaim 1 wherein the patient is a patient suspected of having type IIdiabetes.
 14. The method of claim 13 wherein the type II diabetes isobese type II diabetes, and: a P-type to A-type IPG ratio less thanabout 2 times the ratio in control subjects is indicative of obese typeII diabetes; and an A-type to P-type IPG ratio greater than about 2times the ratio in control subjects is indicative of obese type IIdiabetes.
 15. The method of claim 1, wherein an IPG ratio is determined.16. A method of diagnosing obese type II diabetes, the method comprisingdetermining: (a) the level of A-type inositolphosphoglycans (IPGs), (b)the ratio of P-type to A-type IPGs, or (c) the ratio of A-type to P-typeIPGs in a biological sample from a patient, wherein said P-type IPGs arecapable of activating pyruvate dehydrogenase phosphatase, and saidA-type IPGs are capable of activating lipogenesis in isolatedadipocytes; wherein a level of A-type IPGs or an A-type to P-type IPGratio that is more than about 2-fold higher, or a P-type to A-type IPGratio that is more than about 2-fold lower, than in control subjects isindicative of obese type II diabetes; and wherein the IPG levels aredetermined by immunoassay.
 17. The method of claim 16 wherein thebiological sample is a blood or urine sample.
 18. The method of claim17, the method comprising: (a) contacting the sample with a solidsupport having immobilised thereon a first antibody having one or morebinding sites specific for one or more P-type IPGs and a second antibodyhaving one or more binding sites specific for one or more A-type IPGs;(b) contacting the solid support with labelled developing agents capableof binding to IPGs bound to antibodies or capable of binding to anti-IPGantibodies; and (c) detecting the label of the developing agentsspecifically binding in (b) to obtain values representative of thelevels of the P- and A-type IPGs in the sample.
 19. The method of claim18 wherein (i) at least one of the immobilized first and secondantibodies comprises a monoclonal antibody, (ii) after contact with thesample, the solid support is contacted with a polyclonal antibodycapable of specifically binding to IPGs, and (iii) the developing agentcomprises an antibody capable of specifically binding to the polyclonalantibody.
 20. The method of claim 16 wherein the patient is a patientsuspected of having type II diabetes.
 21. The method of claim 11 whereinthe type II diabetes is obese type II diabetes.
 22. The method of claim16, wherein an IPG ratio is determined.
 23. A method of diagnosing leantype II diabetes, the method comprising determining: (a) the level ofP-type inositolphosphoglycans (IPGs), (b) the ratio of P-type to A-typeIPGs, or (c) the ratio of A-type to P-type IPGs in a biological samplefrom a patient, wherein said P-type IPGs are capable of activatingpyruvate dehydrogenase phosphatase, and said A-type IPGs are capable ofactivating lipogenesis in isolated adipocytes; wherein a level of P-typeIPGs or a P-type to A-type IPG ratio that is more than about 2-foldhigher, or an A-type to P-type IPG ratio that is more than about 2 -foldlower, than in control subjects is indicative of lean type II diabetes;wherein the IPG levels are determined by immunoassay.
 24. The method ofclaim 23 wherein the biological sample is a blood or urine sample. 25.The method of claim 24, the method comprising: (a) contacting the samplewith a solid support having immobilised thereon a first antibody havingone or more binding sites specific for one or more P-type IPGs and asecond antibody having one or more binding sites specific for one ormore A-type IPGs; (b) contacting the solid support with labelleddeveloping agents capable of binding to IPGs bound to antibodies orcapable of binding to anti-IPG antibodies; and (c) detecting the labelof the developing agents specifically binding in (b) to obtain valuesrepresentative of the levels of the P- and A-type IPGs in the sample.26. The method of claim 25 wherein (i) at least one of the immobilizedfirst and second antibodies comprises a monoclonal antibody, (ii) aftercontact with the sample, the solid support is contacted with apolyclonal antibody capable of specifically binding to IPGs, and (iii)the developing agent comprises an antibody capable of specificallybinding to the polyclonal antibody.
 27. The method of claim 23 whereinthe patient is a patient suspected of having type II diabetes.
 28. Themethod of claim 27 wherein the type II diabetes is lean type IIdiabetes.
 29. The method of claim 23, wherein an IPG ratio isdetermined.